Hepatitis B virus genotyping in chronic hepatitis B patients in southwestern Saudi Arabia

braz j infect dis.2015;19(5):525–528

ww w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Brief

communication

Hepatitis

B

virus

genotyping

in

chronic

hepatitis

B

patients

in

southwestern

Saudi

Arabia

Ahmed

Morad

Asaad

_,

_Mohamed

_Saeed

_Zayed

_Al-Ayed

_,

_Mohamed

_Aleraky

_,

Mohamed

Ansar

Qureshi

a_{DepartmentofMicrobiology,CollegeofMedicine,NajranUniversity,Najran,SaudiArabia}

b_{CollegeofAppliedMedicalSciences,DepartmentofPediatric,CollegeofMedicine,NajranUniversity,Najran,SaudiArabia}

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Articlehistory:

Received31December2014 Accepted29March2015 Availableonline18May2015

Keywords: HepatitisB HBVgenotypes GenotypeD Najran

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ThedistributionofhepatitisBvirusgenotypesinSaudiArabiaislargelyunknown.Tothe bestofourknowledgetherearenodataavailableaboutHBVgenotypesinsouthwestern regionofthecountry.Thisstudyaimedtodeterminetheepidemiologicdistributionof hep-atitisBvirusgenotypesinchronichepatitisBpatientsinsouthwesternregion,andtoverify possiblecorrelationsbetweenthesegenotypesandtheclinicalsymptoms.Atotalof160 patientswithchronichepatitisBinfectionwereenrolledinthisstudy.Seraweretested forliverfunctiontests,hepatitisBvirusmarkersandDNAloadbystandardprocedures. HBVgenotypingwasperformedby2-tubenestedPCRfordeterminationofsixgenotypes (A–F).GenotypeDwasthemostcommon,foundin135(84.4%)patients,followedbyA(18; 11.3%)andE(7;4.3%).TherateofHBeAgpositivityingenotypeDpatientswassignificantly lowercomparedwiththatingenotypeAandEpatients(p=0.01).Therewasnosignificant associationbetweenHBVgenotypesandage,gender,liverfunctiontests,orHBVDNAload. GenotypesDandEwerepredominantinchronichepatitisBpatientsinsouthwesternSaudi Arabia.AwarenessofhepatitisBvirusserologicandgenotypicpatternsmighthelpinthe formulationofmanagementplans,predictingclinicaloutcomesandupdatingprevention strategies.

©2015ElsevierEditoraLtda.Allrightsreserved.

Hepatitis B virus (HBV) infection is a potentially life-threateningliverdiseaseandrepresentsamajorglobalhealth problem.About twobillionpeople havebeen infectedwith HBV worldwide, and 400 million among them are suffer-ing from chronic HBV (CHB) infection.1,2 _{In Saudi Arabia,}

the prevalenceof HBV infection hasdeclined considerably sincethe introduction of the HBV vaccine in the national

∗ _{Correspondingauthorat:DepartmentofMicrobiology,CollegeofMedicine,NajranUniversity,P.O.Box1988,Najran,SaudiArabia.}

E-mailaddresses:ahmedmoradasaad@hotmail.com,amasad@nu.edu.sa(A.M.Asaad),drmzayed2000@yahoo.com(M.S.Z.Al-Ayed), dreraki1973@yahoo.com(M.Aleraky),mqansar@yahoo.com(M.A.Qureshi).

immunizationprogramin1989.3 _{However,differentreports}

haveshownthatHBVinfectionscontinuetobeamajorburden ontheSaudihealthcaresystem.AccordingtoAlgarnietal., 23,236casesofHBVinfectionhavebeenreportedtotheSaudi MinistryofHealthduringthe5-yearperiodfrom2009to2013 andtheincidencerateswere19.3and14.7per100,000 popu-lationsin2009and2013,respectively.4

http://dx.doi.org/10.1016/j.bjid.2015.03.007

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According to the molecular evolutionary analysis of genomic DNA sequence, HBV strains isolated in various countries are classifiedinto ten genotypes,designated A–J, andarbitrarilydefinedbyaninter-groupsequencedivergence ofmorethan 8%basedon completegenomes.5,6 _Genotype

distribution shows variationsbetween countries, and even betweengeographicalregionswithincountries. GenotypeA iswidespreadinsub-SaharanAfrica,NorthernEurope,and WesternAfrica;genotypesBandCarecommoninAsia; geno-typeCisprimarilyobservedinSoutheastAsia;genotypeD isdominantinAfrica,Europe,Mediterraneancountries,and India;genotypeEisfoundincentralandwesternAfrica; geno-typeFisdominantinLatinAmericaandAlaska;genotypeG isreportedinFrance,Germany,and theUnitedStates;and genotypeHiscommonlyencounteredinCentralandSouth America.GenotypeIhasrecentlybeenreportedinVietnam and Laos.ThenewestHBV genotype, genotype J,hasbeen identifiedinJapan.6–11

Genotyping of HBV is essential for characterization of patientgroupsandforepidemiologicalstudies.Theclinical significanceofdifferentHBVgenotypeshasbecome increas-inglyrecognizedinpatientswithacuteandCHBinfection.8–10

Moreover,severalstudieshaveshownastrong relationship betweenHBV genotypesandmutationsinthepre-coreand corepromoterregionsthatabolishordiminishtheproduction ofhepatitisBeantigen(HBeAg).ThecourseofHBVinfection dependsonseveralfactorssuchashostgeneticfactors,age andgeneticvariabilityofthevirus.Inadditiontothe epidemi-ologicalimportance,HBVgenotypesmayinfluencethedisease patternandresponsetotreatment.6,12

The distribution of HBV genotypes in the Kingdom of Saudi Arabia is largely unknown, and only a few studies havereportedthepredominanceofHBVgenotypeDinsome locations.13,14_{Tothebestofourknowledgetherearenodata}

availableaboutHBV genotypesinthesouthwesternregion. Hence,theaimsofourstudyweretodeterminethe epidemi-ologicdistributionofHBV genotypesinSaudipatientswith CHBinNajran, acity insouthwesternSaudiArabia,and to verifypossiblecorrelationsbetweenthesegenotypesandthe clinicalsymptoms.

Thiscross-sectionalstudywasconductedatKingKhalid Hospital,a350-bedtertiarycarehospitalinNajranduringa 6-monthperiodfromFebruarytoOctober2014.Atotalof160 patientswithCHBinfection,followed asoutpatientsatthe departmentofinternalmedicinewereenrolledinthisstudy. Sixty-five (40.6%)patients received treatmentwith lamivu-dinemonotherapy.Five (3.1%)patientsreceivedlamivudine followedbyadd-onadefovir,whilenoneoftheremaining90 (56.3%)patients had everreceived antiviral treatment. The inclusioncriteria wereall patientswho wereserum HBsAg positiveforatleastsixmonthswithelevatedALTlevels(>1.5 timestheupperlimitofnormal;65U/L).Allofthepatients werediagnosedaftertheyhadbeenpreviouslyfollowedfor atleast12months.Theexclusioncriteriawerepatientswith acute HAV, HBV, HCV, or HDV, patients with evidence for hepatocellularcarcinoma(HCC),orconcomitantofHCV,HDV, HIVinfection, metastaticor autoimmune liverdisease and druginducedacutehepatitis.Thesocio-demographicdataand laboratorytestsresultswererecorded usingastandardized questionnaire,includingtheage,gender,underlyingmedical

Table1–Characteristicsof160patientswithchronic HBVinfection.

Characteristics Values

Meanage(years) 41.6±4.9

Gender(male/female) 124/36

Totalbilirubin(␮mol/L;normal:3–17) 16.5±1.3

Albumin(g/L;normal:34–50) 39.4±2.5

SerumAST(U/L;normal:15–37) 86.2±32.1

SerumALT(U/L;normal:25–65) 168.7±58.9

HBeAg(positive/negative,n) 74/86

HBVDNAload(log10copies/ml) 15.5±3.4

GenotypeD(n,%) 135,84.4%

GenotypeA(n,%) 18,11.3%

GenotypeE(n,%) 7,4.3%

conditions,signsandsymptomsatpresentation,findingson physicalexamination,laboratoryfindings,anddefinitive clin-icaldiagnosis.

Thelevelsofserumtotalbilirubin,albumin,alanine amino-transferase (ALT) and aspartate transaminase (AST) were determined with routine automated techniques. The HBV markers(HBsAg,HBsAb,HBeAg,HBeAb,HBcAb,andanti-HBc AbIgM)weremeasuredusingtheenzyme-linked immunosor-bent assay(ELISA)kits (RocheLaboratories,Branchburg,NJ, USA).SerumHBVDNAloadinpatientswithCHBwas mea-suredbyasensitivePCRbasedassay(COBASAmplicor,Roche Diagnostics,Branchburg,NJ,USA)withalowerdetectionlimit of approximately 200copies/mL. HBV genotyping was per-formedby2-tubenestedPCR,usingtypespecificprimersfor determinationofsixgenotypesAthroughFofHBVaccording toamethodpreviousdescribedbyFarazmandfaretal.15

Data wereanalyzedusingtheStatisticalPackageforthe Social Sciences (SPSS), Version 15.0 (SPSS Inc., Chicago,IL, USA).Statisticalsignificancewasdefinedasap-valuelessthan 0.05.

Thedemographic,serologicalandvirological characteris-ticsofthepatientsaresummarizedinTable1.Thepatients werebetween32and52yearsofagewithamedianageof41 years.Ofthesepatients,124(77.5%)weremaleand36(22.5%) werefemale.HepatitisBgenotypingrevealedthat135patients (84.4%)hadgenotypeD,18patients(11.3%)hadgenotypeA, and 7patients (4.3%)had genotypeE. Noother HBV geno-typeormixedinfectionsweredetectedinpatientsenrolled forthis study.Thedemographic, serologicalandvirological characteristicswerecomparedamongpatientswiththe dif-ferentgenotypes(Table2).Univariateanalysisindicatedthat therateofHBeAgpositivityingenotypeDpatientswas sig-nificantly lower compared with that in genotype A and E patients(p=0.01).However,therewasnosignificant associ-ationbetweenHBVgenotypesandage,gender,liverfunction tests,orHBVDNAload.

CHB isan importantmedical problem in Saudi Arabia, whichiscurrentlyclassifiedbytheWHOasanareaofhigh endemicity(≥8%ofpopulationareHBsAgpositive).1_Detection

ofHBVgenotypeisveryimportanttoclarifythepathogenesis, routesofinfectionandvirulenceofthevirus.However,there isapaucityofinformationonHBVgenotypepatternsinSaudi Arabia. Inthisstudy,wefoundthatthe majority(84.4%)of patientswereinfectedwithHBVgenotypeD,followedby geno-typesA(11.3%)andE(4.3%).Theseresultsareconsistentwith

brazj infect dis.2015;19(5):525–528

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Table2–Characteristicsof160patientswithchronicHBVinfection,classifiedbyHBVgenotype.

Characteristics HBVgenotype p-Value

D(n=135) A(n=18) E(n=7)

Meanage(years) 42.9+4.9 39.7+4.1 41.4+5.3 0.12

Gender(male/female) 102/33 16/2 6/1 0.28

Totalbilirubin(␮mol/L;normal:3–17) 16.5±1.4 16.4±1.3 16.2±1.2 0.85

Albumin(g/L;normal:34–50) 39±2.1 39.5±2.7 40.4±2.7 0.19

SerumAST(U/L;normal:15–37) 95.9+29.1 73.4+31.6 81.3+32.1 0.06

SerumALT(U/L;normal:25–65) 167.1+59.2 161.2+56.1 193.3+55 0.48

HBeAg(positive/negative,n) 55/80 13/5 3/4 0.01

HBVDNAload(log10copies/ml) 1.2×106 1.8×105 2.3×105 0.25

previousfindingsfromSaudiArabia.InapreviousSaudistudy, 85%of54CHBpatientshadgenotypeD,while5.7%and1.4% hadgenotypesAandE,respectively.14_{Inanotherstudy,Abdo}

etal.showedthat81%,5.7%and1.4%of70hepatitisBpatients hadgenotypesD,E,andA,respectively.13_{Reportsfromthe}

Mediterraneanarea,MiddleEastandcentralandsouthAsia showedthat70–80%ormoreoftheHBVinfectionsarecaused bygenotypeD.7,8,16–19

TheclinicalsignificanceofdifferentHBV genotypes has becomeincreasinglyrecognizedinpatients withacuteand chronicinfection.However,theclinicalimpactofHBV geno-typeDhasbeenstudiedlessextensively.Inthisstudy,similar topreviousreports,age, gender,serum totalbilirubin, liver enzymes,andHBVDNAlevelswerenotsignificantlydifferent betweenpatientsinfectedwithgenotypeDandthoseinfected withgenotypesAorE.9,10 _{Contrary tothesefindings,}

Yous-sifetal.18_{andNabucoetal.}11_{foundasignificantassociation}

betweenALTlevelsandHBVgenotypeswithlower ALT lev-elsobservedingenotypeDpatientsandhigherDNAlevelsin genotypeEpatients.Theclinicaloutcome ofHBV infection andtheseverityofliverdiseasewithgenotypeDandEare stillcontroversial.ItiswellknownthatpatientswithCHBmay havefluctuatingserumALTandHBV-DNAlevels.Further anal-ysesinlarge-scalelongitudinalstudiesarerequiredtobetter delineatethisrelationship.

Inchronic infection,levelsofviremia are generallylow. Long-termprognosisispooreramongHBeAgnegative individ-ualscomparedtotheircounterpartswhoareHBeAgpositive.5

Inthisstudy,asignificantlylowerrateofHBeAgpositivityin patientswithgenotype D wasreported comparedto geno-typeAandEpatients.Inagreement,Yousifetal.18_showed

asignificantlyhigherfrequencyofHBeAgpositivityin geno-type E-infected patients compared to genotype D-infected patients(29.2%versus8.3%,p<0.05).HBeAgnegativeCHBis mostlyassociatedwithmutationsinthepre-coreandbasal corepromoterregionsthatresultinpreventionorreduction ofHBeAgsynthesiswithoutaffectingthereplicativeabilityof thevirus.12,17,19_{Zakyet al.}17 _{foundthat94%of83Egyptian}

patientsinfectedwithHBVgenotypeDwereHBeAgnegative, presumablyindicatingpre-core orcorepromotermutation. Sitniketal.12_{examinedhepatitisgenotypesin103Brazilian}

patients.Pre-coremutantswerefoundin32patients,witha higherfrequencyinthoseinfectedwithgenotypeD(22cases). HBeAgnegativeCHBischaracterizedbyfrequent exacerba-tionofhepatitis,whichprobablyincreasestheriskofcirrhosis, hepaticfailure,andHCC.5_Baigetal.20_{foundthat8of9HCC}

patientshadHBVgenotypeDand4of5cirrhoticpatientshad

HBVgenotypeA.Ourstudydidnotincludepatientswith cir-rhosisorHCC.However,inSaudiArabia,HCCisthesecond mostcommoncancer,3_{andinacountrywhereHBVisendemic}

this could imply acorrelation betweenthe most prevalent genotypeDandHCC.

Thisstudyhadsomelimitations.Firstly,therelativelysmall sample size may have limited our ability to detect differ-enceseventhoughitincludedthehighestnumberofpatients genotypedinSaudiArabia.Secondly,thestudyincludedonly CHBgroupofpatients.Therefore,large-scalestudies includ-ingdifferentHBVclinicalgroupsarerequiredtoobtainfurther informationontheroleofgenotypeDandEandtheirimpact ontheprogressionofliverdiseases.

Inconclusion,ourfindingsfurtherillustrateHBVgenotype patternsinSaudiArabia.GenotypesDandEwere predomi-nantinCHBpatients,andDisthemostprevalentgenotype in Najran,southwestern SaudiArabia. Themajorityof the patientswereHBeAgnegativesuggestingpre-coreorcore pro-motermutations.AwarenessofHBVserologicandgenotypic patternsmighthelpintheformulationofmanagementplans, predictingclinicaloutcomesandupdatingprevention strate-gies.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

Thiswork was supportedbyagrant from theDeanshipof ScientificResearch,NajranUniversity(MID13/26).

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